Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02660507 2015-10-07
. .
Targeted Nanoparticles for Cancer Diagnosis and Treatment
Field of the Invention
This disclosure relates to nanoparticles, and more particularly to targeted
modified gold
nanoparticles for diagnostic and therapeutic applications.
Background of the Invention
Throughout this application, various references are cited in parentheses to
describe more
fully the state of the art to which this invention pertains. Full
bibliographic information for each
citation is found at the end of the specification, immediately preceding the
claims.
Various forms of radiation such as x-rays, laser light, and microwaves, as
well as particle
beams of, for example, neutrons, electrons, and protons, have been used to
treat tumors.
Unfortunately, such radiations are not generally very specific for the tumor
and the dosages used
often results in serious damage to normal tissue, thus limiting irradiation to
lower doses that are not
effective.
Radiosensitizer drugs are also utilized that act in combination with radiation
to produce
improved response, usually by making DNA more susceptible to radiation, or
extending the life of free
radicals produced by the radiation. Other radiation enhancers include elements
or compounds that
interact directly with the radiation to cause more tissue damage by increasing
the absorption or
scattering of the radiation, causing more local energy deposition by
production of secondary
electrons, alpha particles, Auger electrons, ionizations, fluorescent photons,
and free radicals. For
cancer therapy, the purpose is to selectively enhance dose to the tumor, so
these drugs, elements or
compounds must be preferentially accumulated in tumor tissue or the tumor
tissue must respond in a
preferential way, to spare the normal tissue.
Phytodynamic therapy (PDT) utilizes compounds that absorb visible light and
result in
formation of toxic free radicals. However, a disadvantage of this therapy is
that it requires visible
(laser) light to penetrate the tumor and is thus limited to superficial
tissue, or those tissues that are
optically accessible, generally superficial malignancies. Uniformity of dose
delivery is also a problem
due to the high absorbance of the light by tissue.
Boron Neutron Capture Therapy (BNCT) utilizes boron-10-containing compounds
that have a
high cross section for absorption of neutrons. Upon neutron capture, boron
fissions into a lithium ion
and
1
CA 02660507 2009-03-27
alpha particle which have ranges of 5-9 microns and can locally damage DNA and
kill cells. However, this
method has several disadvantages for use.
Metals have been proposed for use in order to reduce cancerous growths. For
example, U.S. Pat.
No. 6,001,054 discloses a method for treating a site in a human body to
inhibit abnormal proliferation of
tissue at the site by introducing a metal surface at the site and then
directing ionizing irradiation to the
metal surface to obtain locally enhanced radiation therapy. Herold et at.
("Gold microspheres: a selective
technique for producing biologically effective dose enhancement," Int. J. Rad.
Biol. 76: 1357-1364, 2000)
discloses the use of gold particles with suspended living cells and with
tumours during irradiation with x-
rays. While a dose enhancement was found, no tumor remission or shrinkage in
the animals was
reported,
While the use of metal nanoparticles for cancer diagnosis and therapy has been
contemplated,
an effective means of how to deliver such nanoparticles to targeted cancer
tissue is required. Various
approaches for targeted delivery have been reported. For example, U.S.
2005/0020869 and U.S.
2005/0256360 disclose the use of gold nanoparticles for administration to
enhance the effects of
radiation therapy.
The advancement of cancer therapeutics, such as precise radiation therapy,
refined
chemotherapy/bio-molecular agents, as well as designer nanotechnology, has
resulted in unprecedented
cancer treatment potentials. Therapeutics in conjunction with traditional
imaging studies that identify
gross anatomy (e.g., computer-assisted tomography), and especially positron
emission tomography (PET)
has become an important clinical practice that detects metabolism and biology
of cancer prior to gross
tumor visibility.
it would be advantageous to provide a nanoparticle technology for cancer
diagnosis and therapy
that can be used in conjunction with imaging technology in an advantageous
manner compared to that of
the prior art.
Summary of the Invention
The present invention provides targeted modified gold nanoparticles (GNPs)
wherein the
modification to the GNPs leads to the effective targeting of the particles to
a desired tissue for the
provision of early diagnosis, imaging and/or treatment of diseases such as
cancers. The present invention
also includes the use of such GNPs in compositions and methods for the
imaging, diagnosis and/or
treatment of diseases.
In an aspect of the present invention is a modified gold nanoparticle
comprising a gold core and a
surface thereon, wherein said surface comprises a modification selected from a
coating of cysteamine
and/or cysteamine/thioglucose.
2
CA 02660507 2009-03-27
In an aspect of the present invention there are provided methods of
eliminating tissue or cells by
delivery of modified gold nanoparticles to the tissue or cells, then applying
external energy that interacts
= =
with the modified gold nanoparticles.
In further aspects, the present invention provides methods of enhanced
radiation therapy for
promoting the shrinkage and/or elimination of tissues targeted for destruction
by using targeted modified
gold nanoparticles.
According to another aspect of the invention there are provided modified gold
nanoparticles that
enable a non-invasive, real time, targeted cancer imaging-therapeutic in one
step. After reaching the
cancer targets, the designer nanoparticles significantly enhance conventional
treatment modalities at the
cellular level. In this aspect the gold nanoparticles of the invention are
modified to be bound to a Positron
Emission Tomography (PET) tracer.
According to another aspect of the present invention are modified gold
nanoparticles comprising
a gold core and a surface thereon, wherein said modification comprises a
coating of cysteamine or
cysteamine/thioglucose on said surface. The coating may be provided as
separate layers on said gold
core.
According to an aspect of the present invention is a targeted modified gold
nanoparticle
comprising a gold nanoparticle bound to a PET tracer.
According to another aspect of the present invention is a targeted modified
metal nanoparticle
comprising a metal nanoparticle bound to a PET tracer.
According to another aspect of the present invention is a targeted modified
gold alloy
nanoparticle comprising a gold alloy nanoparticle bound to a PET tracer.
According to another aspect of the present invention is a targeted modified
gold nanoparticle
comprising a gold nanoparticle covalently bound to a PET tracer.
According to another aspect of the present invention is a targeted modified
gold nanoparticle
comprising a gold nanoparticle bound to a PET tracer, wherein said PET tracer
is selected from an organic
molecule labeled with a radionuclide selected from 11carbon, unitrogen,
18oxygen and 18fluorine.
In aspects the PET tracer can be selected from [18F]flurodeoxyglucose and
[18F]fluro-17-
estradiol.
In aspects of the invention the modified gold nanoparticle comprises a gold
nanoparticle, a
hollowed gold nanoparticle, a gold nanoparticle having a functional molecule
attached thereto, and a
hollowed gold nanoparticle having a functional molecule attached thereto. In
aspects the functional
molecule is selected from cysteamine (AET) and thioglucose (Glu).
According to another aspect of the present invention there is provided a
targeted modified gold
nanoparticle comprising a gold nanoparticle covalently bound to a PET tracer,
wherein said PET tracer is
selected from 118Fiflurodeoxyglucose and [18F]fluro-17-estradiol. In further
aspects, the gold
3
CA 02660507 2009-03-27
v
nanoparticle is hollowed and/or comprises AET and/or Glu.
According to a further aspect of the present invention is a composition
comprising targeted
modified gold nanoparticles.
According to another aspect of the present invention there is provided a
composition comprising
a gold nanoparticles covalently bound to a PET tracer, wherein said PET tracer
is selected from
[18FJfiurodeoxyglucose and [18Fjfiuro-17-estradiol. In further aspects, the
gold nanoparticle is hollowed
and/or comprises AET and/or Glu.
According to another aspect of the present invention there is provided a
method for real time
targeted cancer imaging/therapy, said method comprising administering to a
mammal diagnosed with a
cancer, an effective amount of a modified gold nanoparticle covalently bound
to a PET-tracer and
subjecting said mammal to positron emission tomography for a time effective to
visualize said cancer.
=According to another aspect of the present invention is a method for
enhancing the effects of
radiation directed to a tissue or a population of cells in a mammal,
comprising administering an amount of
targeted modified gold nanoparticles to said mammal to achieve a concentration
in said tissue or said
population of cells of the mammal of at least about 0.1% metal by weight; and
subsequently irradiating
the mammal with radiation directed to said tissue or said population of cells,
wherein said radiation is in
= the form of x-rays of about 1 keV to about 25,000 keV.
The present invention also provides methods for enhancing the effects of
radiation directed to a
tissue or cells in or from a mammal by administering an amount of targeted
modified gold nanoparticles
of the invention to the mammal or to the tissue or cells ex vivo, then
irradiating the mammal with
radiation directed to the tissue or cells, or irradiating the tissue or cells
ex vivo. The methods of the
present invention are useful for ablating unwanted tissues in a mammal without
unacceptable damage to
surrounding normal tissues or substantial toxicity to the mammal.
The present invention also provides methods for imaging tissue or cells in or
from a mammal by
administering an amount of targeted modified gold nanoparticles of the
invention to the mammal or to
the tissue or cells ex vivo, then applying PET scanning techniques to image
the mammal or the tissue or
cells ex vivo.
The present invention also provides methods for diagnosis and treatment of
tissue or cells in or
from a mammal by administering an amount of targeted modified gold
nanoparticles of the invention to
the mammal or to the tissue or cells ex vivo, then applying PET scanning
techniques to image the mammal
or the tissue or cells ex vivo for the purpose of diagnosis and/or treatment.
According to further aspects of the invention are methods of making the
targeted modified gold
nanoparticles of the invention.
According to yet another aspect of the invention is a method of making a
targeted modified gold
nanoparticle, wherein said method comprises;
4
CA 02660507 2015-10-07
- covalently attaching a PET tracer to a gold nanoparticle, wherein said PET
tracer is selected
from an organic molecule labeled with a radionuclide selected from "carbon,
13nitrogen, 15oxygen and
18fluorine. In aspects of the method, the PET tracer can be selected from
[18F]flurodeoxyglucose and
[189fluro-17-estradiol. In further aspects of the method, the gold
nanoparticle can have a having a
functional molecule attached thereto. In aspects the functional molecule is
selected from cysteamine
(AET) and thioglucose (Glu).
In another aspect, the custom-designed gold nanoparticles (in stable binding
with the PET-
avid tracer) enable a non-invasive, real time, targeted cancer imaging-
therapeutic in one step, by
combining (i) enhanced imaging, (ii) radiotherapy, and (iii) real-time imaging
in one single platform. In
one embodiment, the PET-guided gold nanoparticles of the present invention
combine
[189flurodeoxyglucose for imaging and gold nanoparticles (GNPs) for treatment.
After reaching the
cancer targets, the designer nanoparticles significantly enhance conventional
treatment modalities at
the cellular level. When an external irradiation source, like X-ray, strikes
the GNPs, radicals are
generated to induce DNA damage. Due to synergistic multi-level targeting, the
therapy can be
effective in treating breast cancer with minimal in vivo side-effects to the
normal tissue.
In another aspect, radiotherapy of breast cancer cells using the gold-based
nanoparticles
immuno-targeted to molecular markers on the cell surface is an effective
modality to selectively kill
cancer cells. The localized DNA damage can cause the rupture of the targeted
cancer cell membranes
and induce cell apoptosis. In MCF-7 breast adenocarcinoma cells, it engages
the intrinsic pathway by
enhancing upstream caspase activation. Radicals can be generated when X-ray
strikes gold
nanoparticles.
In another aspect, the present invention has the following advantages: 1)
feedback
information from pathology biomarkers can be used for the feature selection of
functional targeting,
2) currently available 3D conformal radiotherapy planning can direct
irradiation precisely at any depth
inside body, and 3) synergistic radiation effect can be achieved at cellular
level.
Other advantages of the present invention include:
i) Active and specific binding will significantly increase the local
concentration of GNPs in
cytoplasm. As a result, GNPs in the cytoplasm kill cancer cells more
efficiently than those on the cell
membrane and are a better choice for X-ray radiotherapy.
ii) Glu-GNPs enhance the radiation sensitivity in cancer cells, but not in
nonmalignant cells.
Therefore, lower irradiation dose is needed and thus reduce side effects of
many cancer patients after
radiotherapy.
iii) Synergistic cancer diagnosis and treatment can be achieved at a cellular
level.
In another aspect, there is provided a pharmaceutical composition comprising
modified gold
nanoparticles, said modified gold nanoparticles comprising:
a gold core; and
CA 02660507 2015-10-07
, .
a surface thereon, wherein said modification comprises a coating of cysteamine
and/or
thioglucose covalently bound to a surface of said core and externally exposed
to target a mammalian
cancer cell.
Other features and advantages of the present invention will become apparent
from the
following detailed description. It should be understood, however, that the
detailed description and
the specific examples while indicating embodiments of the invention are given
by way of illustration
only, since
5a
CA 02660507 2015-10-07
various changes and modifications within the scope of the invention will
become apparent to those
skilled in the art from said detailed description.
Description of the Figures
The present invention will be further understood from the following
description with
reference to the Figures, in which:
Figures 1A and 1B. Gold nanoparticles: 1A. Electric micrograph of gold
nanoparticles (Bar =
50 nm); and 1B. the schematic diagram of naked gold nanoparticle binding with
glucose.
Figure 2. DU-145 cell uptakes of GNPs. Data are reported as the mean SEM for
three
separate experiments performed in quadruplicate. The cell uptake values were
(2.01 0.25)x104/cell
for TGS-GNPs and (6.73 0.69) x104/ cell for GNP-Glu. P < 0.005 for GNP-Glu vs
GNPs (t test).
Figure 3 shows the distribution of GNPs in DU-145 cells.
Figure 4. DU-145 cell growth was decreased by 13.52%, 17.82% and 14.72% after
the
treatment with TGS-GNPs, GNP-Glu, or 2 Gy X¨ray at 24h, respectively. Cell
growth was not different
after exposure to TGS-GNPs, GNP-Glu or 2 Gy X-ray treatment in vitro (P>0.1).
Figures 5A and 5B. Nanoparticles enhanced significantly radiosensitivity of DU-
145 cells.
Irradiation of 2Gy alone induced the inhibition rate of cell growth
(15.78+2.85)% after 24 hours and
(19.03+6.00)% after 48 hours. A. Irradiation of 2Gy+TGS-GNPs were
(30.57+3.32)% at 24 hours and
(32.18+2.12)% at 48 hours. B. Irradiation of 2Gy+Glu-GNPs were (45.97+3.95)%
at 24 hours and
(44.63+1.87)% at 48 hours. Both TGS-GNPs and Glu-GNPs can increase
radiosensitivity of 2Gy (P <
0.001) X-ray on DU-145 cells after 24 and 48 hours.
Figure 6. Comparison of radiosensitivity enhancements of between DU-145 cells
induced by
Glu-GNPs and those induced by TGS-GNPs. The combination of Glu-GNP with X-ray
induced an
increased inhibition rate of 50.3% and 38.7% compared with GNPs only after 24h
(P<0.005), and 48h
(P<0.001) respectively.
Figure 7. Schematic diagram of synthesis of thioglucose-capped gold
nanoparticle (Glu-
Capped GNPs), and Cysteamine-capped gold nanoparticles (AET-capped GNPs).
6
CA 02660507 2009-03-27
Figure 8. (A) TEM and (B, C) HRTEM images of GNPs with the diameter about 10.8
nm (refer
to inset A). The size distribution of GNPs was determined by DLS.
Figure 9. XPS results of (A) AET-GNPs and (B) Glu-GNPs.
Figure 10. TEM images of MCF-7 cell uptakes for GNPs, the targeted cells were
treated with or
without GNPs for two hours: (A) Control cell; (B) AET gold nanoparticles; and
(C) Glucose gold
nanoparticles. Left bottom subfigure shows an enlarged picture of the part in
Figure 10 (C).
Figure 11. Cell uptakes of GNPs bound with either AET or thioglucose.
Figure 12. Toxicity test of AET-GNP, Glu-GNP and control in MCF-7 cells by MTT
assay.
Figure 13. (A) The cytotoxicity on MCF-7 breast cancer cells induced by 200
kVp X-ray irradiation
with or without gold nanoparticles at 48 hours. (B) Cell survival rate
determined by clonogenic survival
assay (at 5 days and 14 days).
Figure 14. A. Comparison of radiation sensitivity in cancers and non-malignant
cells. B.
Comparison of the cytotoxicity induced in MCF-7 cell line by various types
irradiation with gold
nanoparticles (48 hours after irradiation).
Figure 15 shows the schematics of PET-guided Gold Bullet and the
[18F]flurodeoxyglucose
molecule.
Figure 16. (A) TEM and (B, C) High-resolution TEM images of GNPs with the
diameter about 10.8
nm. (insert of A) The size distribution of GNPs determined by DLS.
Figure 17. NMR spectrum to validate the covalent binding between nanoparticles
and PET
tracers. Here Ac (Acetyl) is a functional group and is used to protect the
binding site. Ac0 will is replaced
by OH group when it is to be used as FDG.
Figure 18. TEM images of MCF-7 cell uptakes for GNPs, the targeted cells were
treated with or
without GNPs for two hours: (A) Control cell; and (C) Glucose gold
nanoparticles. Left bottom subfigure
shows an enlarged picture of the part in Figure 18(C).
7
CA 02660507 2009-03-27
Figure 19. Comparison of MCF-7 cell tumour volume in mice treated with control
(-IN-); FDG-
GNPs (-.-); irradiation only (-A.); and irradiation plus FDG-GNPs
Detailed Description of the Invention
Unless defined otherwise, all technical and scientific terms used herein have
the meaning
commonly understood by a person skilled in the art to which this invention
belongs. As used herein, the
following terms have the meanings ascribed to them unless specified otherwise.
As used herein, "positron emission tomography imaging (PET)" incorporates all
positron emission
tomography imaging systems or equivalents and all devices capable of positron
emission tomography
imaging. The methods of the invention can be practiced using any such device,
or variation of a PET device
or equivalent, or in conjunction with any known PET methodology. See, e.g.,
U.S. Pat. Nos. 6,151,377;
6,072,177; 5,900,636; 5,608,221; 5,532,489; 5,272,343; 5,103,098. Animal
imaging modalities are
included, e.g. micro-PETS (Corcorde Microsystems, Inc.).
As used herein, external energy, e.g., radiation, is directed to a tissue or a
population of cells
targeted for destruction or ablation, also referred herein as a "target
tissue" or "target cells". By "ablating
a tissue or cell" is meant that the growth of the tissue or cell is inhibited,
the size of the tissue or the
number of the cells is reduced, or the tissue or cell(s) is eliminated. By
administering metal nanoparticles
to a mammal, or to tissues or cells ex vivo, the therapeutic effects of
external energy or radiation is
enhanced by way of the interaction of the particles with the sources of energy
or radiation, resulting in an
increased energy deposition in the vicinity of the particles. This is also
referred to herein as "Metal
Enhanced Radiation Therapy", or "MERT". Metal-Enhanced Radiation Therapy
(MERT) is able to kill
= unwanted tissue or cells in a highly specific manner.
As used herein, "enhanced radiation therapy" or "enhancing the therapeutic
effects of radiation"
is meant that a lower dose of radiation is required to achieve efficacy (e.g.,
the target tissue is ablated or
eliminated) with metal nanoparticles as compared to without metal
nanoparticles; or, better efficacies are
achieved by a given dose of irradiation with metal nanoparticles as compared
to without metal
nanoparticles.
Targeted Gold Nanooarticles
The present invention provides gold nanoparticles (GNPs) for use in the
imaging, diagnosis
and/or treatment of disorders in vivo and ex vivo. The gold nanoparticles may
be modified with different
functional molecules such that they target cells in vivo or ex vivo in mammals
and mammalian tissues.
The gold nanoparticles may also be modified to be hollow in order to
encapsulate a drug and act as a
controlled release drug carrier. The gold nanoparticles of the invention may
also further be bound to a
8
CA 02660507 2009-03-27
PET-tracer to enable cancer imaging and therapeutics with radiation and in
particular, the targeted gold
nanoparticles of the invention are used to image, diagnose and treat various
forms of cancers in a
mammal.
Modifications to the GNPs of the invention include but are not limited to
providing different
functional molecules as a coating thereon. The functional molecules may be
selected from a type of
amine coating such as cysteamine (AET), thioglucose (Glu) and combinations
thereof. As modified using
such functional amines, the gold nanoparticles may bind to the outside of
cells (cysteamine) or
intracellularly (Glu). AET-capped GNPs are strongly positive and selectively
bind onto the cell's surface.
Glu-capped GNPs target the cell cytoplasm and take advantage of the fact that
cancer cells have an
increased requirement for glucose. Such modified GNPs enhances radiation
cytotoxicity and provides a
more effective cancer treatment.
Radiation can be in the form of low energy (about 1 to 400 KeV) or high energy
x-ray (about 400
KeV up to 25,000 KeV). Radiation can also be in the form of microbeam arrays
of x-ray or radioisotopes.
Other forms of radiation suitable for use in practicing the methods of the
present invention include, but
are not limited to, visible light, lasers, infrared, microwave, radio
frequencies, ultraviolet radiation, and
other electromagnetic radiation at various frequencies. Various sources or
forms of radiation can be
combined, particularly for treating tumors at depth.
Enhanced radiation therapy may be applied to a human to destroy unwanted
tissue, e.g., a
tumor. The modified gold nanoparticles can be administered to human (or
animal) prior to irradiation by
standard methods, e.g., intravenous or intra-arterial injection, direct
injection into a target tissue (e.g.,
tumor), and implantation of a reservoir device capable of a slow release of
the modified gold
nanoparticles. In general, nanoparticles are administered in an amount to
achieve a concentration in the
human/animal of at least about 0.05 to 10% gold by weight, preferably 0.1 to
5% gold by weight, and
more preferably 0.3% to 2% gold by weight, in order to achieve radiation
enhancement. The enhanced
radiation therapy may be applied to tissues or cells ex vivo to ablate or
destroy unwanted tissues or cells
from a human/animal. For example, the enhanced radiation methods can be
applied to bone marrow ex
vivo to eliminate unwanted cells prior to transplantation, or applied to a
donor organ to remove
immunogenic cells prior to transplantation.
The enhanced radiotherapy methods disclosed herein can be used in conjunction
with other
existing therapies, such as chemotherapy, anti-angiogenesis therapy, boron
neutron capture therapy (or
BNCT) and other drug therapy.
The nanoparticles disclosed herein can also be used as drug delivery agents.
Due to the size of
the nanoparticles, the use of nanoparticles to deliver drugs to the brain
across the blood-brain barrier
(BBB) is possible. An advantage of nanoparticle carrier technology is that
nanoparticles mask the blood-
brain barrier limiting characteristics of the therapeutic drug molecule. The
system may slow drug release
9
CA 02660507 2009-03-27
in the brain, decreasing peripheral toxicity. Thus, the nanoparticles
disclosed herein can be used in non-
limiting aspects to treat Central Nervous System (CNS) diseases. The PET-
guided nanoparticles can also
access brain metastasis in cancers.
In non-limiting embodiments of the invention, modified gold nanoparticles
(GNPs) were
developed and synthesized with two kinds of functional molecules: Cysteamine
(AET) and thioglucose
(Glu). Their cell uptake and radiation cytotoxicity enhancement in a breast
cancer cell line (MCF-7) versus
a non-malignant breast cell line (MCF-10A). Transmission Electron Microscopy
(TEM) results showed that
cancer cells take up functional Glu-GNPs significantly more than naked GNPs.
The TEM results also
indicated that AET-capped GNPs are mostly bound to the MCF-7 cell membrane,
while Glu-GNPs enter the
cells and are distributed in the cytoplasm. After MCF-7 cell uptake of Glu-
GNPs, the in.vitro cytotoxicity
effects were observed at 24, 48, and 72 hours. The results showed that these
functional GNPs have little
or no toxicity to these cells. To validate the enhanced killing effect on
cancer cells, we have applied
various forms of radiation, such as 200kVp X-rays and y-rays, to the cells,
both with and without functional
GNPs. By comparison with irradiation alone, the results showed that GNPs
significantly enhanced cancer
killing.
Prostate Cancer ¨ Modified Gold Nanonarticles Enhanced Radiation Sensitivity
in Prostate Cancer Cells
Gold nanoparticles (GNP) were made and used to enhance radiation sensitivity
and growth
inhibition in radiation-resistant human prostate cancer cells. Gold
nanoparticles (GNPs) were
synthesized as described herein in Example One. Exposure to Glu-GNPs resulted
in a three times
increase of nanoparticle uptake compared to that of TGS-GNPs in each target
cell (p<0.005). Cytoplasmic
intracellular uptake of both TGS-GNPs and Glu-GNPs resulted in a growth
inhibition by 30.57% and 45.97%
respectively, comparing to 15.88% induced by irradiation alone, in prostate
cancer cells after exposure to
the irradiation. GluGNPs showed a greater enhancement, 1.5 to 2 fold increases
within 72 hours, on
irradiation cytotoxicity compared to TGS-GNPs. Tumour killing, however, did
not appear to correlate
linearly with nanoparticle uptake concentrations. These results showed that
functional glucose-bound
gold nanoparticles enhanced radiation sensitivity and toxicity in prostate
cancer cells.
Breast Cancer ¨ Modified Gold Nanooarticles Enhanced Radiation Cvtotoxicitv in
Breast Cancer Cells
Gold nanoparticles (GNPs) and modified GNPs with two kinds of functional
molecules:
Cysteamine (AET) and thioglucose (Glu) were synthesized and demonstrated their
cell uptake
and radiation cytotoxicity enhancement in a breast cancer cell line (MCF-7)
versus a non-
malignant breast cell line (MCF-10A). Transmission Electron Microscopy (TEM)
results showed
that cancer cells take up functional Glu-GNPs significantly more than naked
GNPs. The TEM
results also indicated that AET-capped GNPs were mostly bound to the MCF-7
cell membrane,
CA 02660507 2009-03-27
while GluGNPs enter the cells and are distributed in the cytoplasm. After MCF-
7 cell uptake of
Glu-GNPs, or binding of AET-GNPs, the in vitro cytotoxicity effects were
observed at 24, 48,
and 72 hours, respectively. The results showed that these functional GNPs have
little or no toxicity
to these cells. To validate the enhanced killing effect on cancer cells,
various forms of radiation,
such as 200kVp X-rays and y-rays, to the cells, both with and without
functional GNPs. By
comparison with irradiation alone, these results showed that GNPs
significantly enhanced cancer
killing.
As described above gold nanoparticles can be made to be solid or hollow to
contain a
desired therapeutic drug. The gold nanoparticles can also be made to have
different functional
molecules as a coating thereon as is described herein. The functional
molecules can be coated on gold
nanoparticles that are either solid or hollow. If hollow the gold
nanoparticles can be provided with the
functional molecules as a coating and also contain a drug within the gold
nanoparticle.
The gold nanoparticles of the invention for use in radiation therapy are
composed of a metal core
and provided with a modified surface layer surrounding the metal core. The
metal core in many aspects
of the invention is gold. However, it is also understood by one of skill in
the art that the metal may be
made of a gold alloy or other metal altogether. Metals which can be used to
form the modified targeted
nanoparticles of the invention for enhancing radiation effects are heavy
metals, or metal with a high Z
number, including but not limited to gold, silver, platinum, palladium,
cobalt, iron, copper, tin, tantalum,
vanadium, molybdenum, tungsten, osmium, iridium, rhenium, hafnium, thallium,
lead, bismuth,
gadolinium, dysprosium, holmium, and uranium. The preferred metal is gold in
an aspect. The metal core
can consist of one metal, or it can be a mixture or an ordered, concentric
layering of such metals, or a
combination of mixtures and layers of such metals.
The size of the gold core may be from about 0.8 up to about 400 nm in
diameter. The gold
nanoparticles of this size selectively increase the local radiation dose
directed to a target tissue such as a
tumor and do not exhibit substantial toxicity in the mammal. Preferably, the
core is of a size in the range
of at least about 0.8 or 1 to 50 nm; more preferably, about 0.8 or 1 to 20 nm
in diameter; and even more
preferably, about 0.8 to 3 nm in diameter. Especially preferred size of the
metal core is up to about 10-11
nm in diameter.
In all embodiments of the gold nanoparticles described herein, the modified
gold nanoparticle
may be bound to a PET tracer in order to be used for early cancer diagnosis
and the modification may be
used effectively to provide therapy. In aspects, this is covalent binding. The
PET tracer may be selected
from an organic molecule labeled with a radionuclide selected from licarbon,
13nitrogen,15oxygen and
13fluorine. In aspects the PET tracer can be selected from
[18F]flurodeoxyglucose and [18F]fluro-17-
estradiol.
In one non-limiting embodiment of the present invention the gold nanoparticle
of the invention
11
CA 02660507 2009-03-27
is covalently bound to [18F]flurodeoxyglucose and is used for early cancer
diagnosis. In aspects the cancer
is breast cancer and prostate cancer. Because of the flurodeoxyglucose, the
gold nanoparticles can be
specifically targeted at tumor cells but not normal cells.
In aspects of the invention the tissues or cells targeted for destruction are
cancerous (i.e. tumors)
and include any solid tumors such as carcinomas, brain tumor, melanomas,
lymphomas, plasmocytoma,
sarcoma, glioma, thymoma, and the like. The present invention can also be used
to treat tumors of the
oral cavity and pharynx, digestive system, respiratory system, bones and
joints, soft tissue, skin, breast,
genital system, urinary system, prostate, eye and orbit, brain and other
nervous system, endocrine
system, myeloma, and leukemia.
Formulation and Administration Pharmaceuticals
The invention provides pharmaceutical formulations comprising the targeted
modified gold
nanoparticles (GNPs) of the invention and a pharmaceutically acceptable
excipient suitable for
administration an imaging enhancing agents, and methods for making and using
these compositions.
These pharmaceuticals can be administered by any means in any appropriate
formulation. Routine means
to determine drug regimens and formulations to practice the methods of the
invention are well described
in the patent and scientific literature. For example, details on techniques
for formulation, dosages,
administration and the like are described in, e.g., the latest edition of
Remington's Pharmaceutical
Sciences, Maack Publishing Co, Easton Pa. As used herein, a pharmaceutically
acceptable carrier includes
solvents, dispersion media, isotonic agents and the like. Examples of carriers
include water, saline
solutions, sugar, gel, porous matrices, preservatives and the like, or
combinations thereof.
The modified gold nanoparticles of the invention may be administered to an
animal by standard
methods, including but not limited to intravenous or intra-arterial injection,
direct injection into a target
tissue (e.g., tumor), and implantation of a reservoir device or cavity capable
of a slow release of metal
nanoparticles. Intravenous injection is well suited to delivery of gold
nanoparticles to the vascular system
of an animal, and is a preferred method of administration where the target
tissue to be ablated is a =
tumor.
Direct intratumoral or tissue injection is also possible in order to reduce
the concentration of
metal nanoparticles in other tissues and achieve a high concentration in the
tumor or tissue to be ablated.
Use of small gold nanoparticles, e.g., 0.8 to 10 nm in diameter, facilitates
diffusion of the particles
throughout the parenchyma of the targeted tissue. Linking nanoparticles to a
specific targeting molecule
(i.e. PET tracer) can facilitate localization of the nanoparticles to specific
cells, as disclosed hereinabove.
A reservoir of the modified gold nanoparticles can be provided adjacent to or
in the bed of a
target tissue. The reservoir can be a permeable bag or container, or a wafer,
string, gel, Matrigel, or other
material preloaded with the nanoparticles, or a time release pump driven
osmotically, mechanically, or
12
CA 02660507 2009-03-27
electrically. The modified gold nanoparticles can be delivered over a
controlled time period, and longer
exposure to the nanoparticles may be achieved as compared to administration by
intravenous injection.
The pharmaceutical formulations of the invention comprising the targeted
modified gold
nanoparticles (not having the PET tracer) can be presented in unit-dose or
multi-dose sealed containers,
such as ampoules and vials, and can be stored in a freeze-dried (lyophilized)
condition requiring the
addition of the sterile liquid excipient, for example, water, for injections,
immediately prior to use.
Extemporaneous injection solutions and suspensions can be prepared from
sterile powders, granules, and
tablets.
It is understood by one of skill in the art that the targeted modified gold
nanoparticles of the
invention can be provided in a formulation in a combination of differently
made nanoparticles. That is a
combination of AET-coated GNPs and Glu-coated GNPs and/or GNPs-PET tracer can
be used together in a
formulation or used concurrently as desired.
Kits
The invention provides kits comprising the targeted modified gold
nanoparticles of the invention
and compositions containing such. The kits also can contain instructional
material teaching
methodologies, e.g., how and when to administer the pharmaceutical
compositions, how to apply the
compositions and methods of the invention to imaging systems, e.g., computer
assisted tomography
(CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging
(MRI), positron emission
tomography (PET) or single-photon emission computed tomography (SPECT). Kits
containing
pharmaceutical preparations may include directions as to indications, dosages,
routes and methods of
administration, and the like.
The above disclosure generally describes the present invention. A more
complete understanding
can be obtained by reference to the following specific Examples. These
Examples are described solely for
purposes of illustration and are not intended to limit the scope of the
invention. Changes in form and
substitution of equivalents are contemplated as circumstances may suggest or
render expedient. Although
specific terms have been employed herein, such terms are intended in a
descriptive sense and not for
purposes of limitation.
13
CA 02660507 2009-03-27
Examples
Example One - Enhanced Radiation Sensitivity in Prostate Cancer by Gold-
Nanoparticles
Chemicals
All chemicals were obtained from Sigma¨Aldrich (Milwaukee, WI). MTT CellTiter
96 non-
radioactive cell proliferation assay kit was purchased from Promega (Madison,
WI).
Synthesis of Gold Nanoparticles
The general synthesis method for making gold nano-particles follows three
substeps. 0 3.2
ml of 25mM HAuC14 solution was added into 60 ml of deionized water in an ice
bath with
moderate stirring. ii) 4 ml of 26 mM NaBH4 was then added as a reductant to
obtain naked gold
nanoparticles. iii) The naked GNPs solution was added into two tubes each
containing 22.4 ml
of naked GNPs solution. 4 ml of 20 mM 1- thio-13-glucose or 4 ml of 38.8 mM
sodium citrate solution was
added separately into two gold solutions.
Thio-glucose covalently and sodium citrate electrostaticly bind to the GNPs to
form
functionalized thioglucose-capped gold nanoparticles (Glu-GNPs) and neutral
gold nanoparticles
(TGS-GNPs) respectively. (Figure 1A and 1B). Both the TGS-GNPs and the Glu-
GNPs were dialysed for
two days to remove any free sodium citrate or thio-glucose from the gold
particle solutions before
these solutions were provided to the experiments. Both the TGS-GNPs and the
Glu-GNPs were
characterized using transmission electronic microscopy (TEM), 1CP-MS and
Kratos Axis 165 X-
ray Photoelectron Spectroscopy (XPS) (Kratos Analytical) as described
previously."
Cell Culture
Human prostate carcinoma cell line DU-145 was used in all the experiments. DU-
145 cell
line was purchased from the American Type Culture Collection (Manassas, VA,
USA). The cells were
maintained in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 20
mM D-glucose,
100 U1/m1 penicillin G, and 100 gg/ml streptomycin in a humidified incubator
with 5% CO2 in the air
at 37*C. DMEM without glucose was used for the cells that were exposed to
either Giu-GNPs or
Giu-GNPs plus Cytochalasin B (glucose transport inhibitor).
Uptake Assay
The assay was performed in triplicate. A 10m1DU145 cell suspension containing
2 x 106 cells
was seeded onto a 100mm-cell culture dish and was cultured overnight. When the
cells
14
CA 02660507 2009-03-27
reached a 70% confluence, the target cells were exposed to the vehicle, 15nM
TGS-GNPs, or 15 n M
GI u-G N Ps, respectively at 37 C. After two hours of incubation, the free
GNPs in the cell cultures
were removed by washing the cells twice with the PBS buffer. The cells were
detached with
Trypsin-EDTA. After centrifugation and the removal of the supernatant, the
cells were
resuspended in the PBS with a final volume of 5 ml. The number of cells in
suspension was counted
with a hemocytometer. 5 ml of 50% HNO3 was added to each sample to lyse the
cells. The gold mass
in the lysis solution was measured using Inductively Coupled Plasma Mass
Spectrometry (ICP-MS). The
number of gold nanoparticles was calculated via the gold mass, and the number
of GNPs in the lysis
solution was divided by the number of cells to yield the number of GNPs taken
up by cells.
Transmission electron microscoov
The cells treated with or without GNPs were collected by centrifugation. The
cell pellets
were fixed in 4% (v/v) formaldehyde in 0.1 M phosphate buffer (pH 7.2) for
four hours at 4 C.
After being washed in the same buffer, the cells were resuspended in 1% 0s04
for one hour at
room temperature. They were then washed twice by centrifugation and
resuspended in
distilled water. The final pellet was resuspended in a small volume of warm 2%
(w/v) agarose,
poured onto a glass slide, and allowed to cool. When set, the small pieces of
gel containing the cells were
cut out and dehydrated through a graded series of ethanol solutions. The
pieces were then
embedded in epoxy resin, and thin sections were cut with an ultramicrotome,
stained with uranyl
acetate followed by lead citrate and examined in Philips EM301 electron
microscope operating at
80 kV.
Irradiation of Cells
All cell irradiation treatments were carried out using a Pautak Therapax 3
Orthovoltage 244
Monitor Units /minute X-ray machine at 200 kVp using a 0.35 CU + 1.5 AL
filter. 0U145 cells in 100-
mm culture dishes were irradiated at room temperature when cultures reached
75%
confluence. Cells received either a mock treatment for control or 2 Gy. After
irradiation, cultures
were returned to 5% CO2/37 C incubation until they were harvested at the time
points required.
MTT Assay
MIT assay is a quantitative colorimetric method to determine cytotoxicity. It
utilizes the
yellow tetrazolium salt [3-(4,5-Dimethylthiazol-2-y1)-2,5- diphenyltetrazolium-
bromide] which is
metabolized by mitochondrial succinic dehydrogenase activity of proliferating
cells to yield a purple
formazan reaction product. In the present study, toxicity in mitochondria
induced by GNPs with or
without radiation was measured by an MTT assay in 96-well plates. The
experiments were
CA 02660507 2009-03-27
performed by eight replicates and cells were seeded in 96-well plates
(3x103/well) with 200 IA of
culture medium per well. The cells were allowed to grow on the 96-well plates
overnight. Cells
were then exposed to either TGS-GNPs or Glu-GNPs respectively and different
doses of irradiation
according to the experimental design. The cell responses were monitored by the
MTT assay by following
the manufacturer's instructions. After removal of the medium, 100111 of MTT
(0.4 mg/ml) dissolved in
medium were added to each well. Following three hours of incubation, the
medium was replaced
with 100 of 0.1 N HC1/isopropanol, and absorbance in each well was assessed at
550 nm using a
microplate reader. Absorbance was expressed as a percentage of control. The
cell growth
inhibitory rate was calculated by the following formula: Inhibitory rate = (1-
average OD550nrn of treated
group/average ODssonm of control group) x100%.
Statistical Analysis
In the statistical analysis, differences between the treated and control
groups were
compared using Student's t-tests, with the differences at the P<0.05 level
considered to be statistically
significant.
Uptake of GNPs on DU-145 cells
After exposure to 15 nM naked TGS-GNPs and Glu-GNPs respectively for two
hours, the
average number of GNPs per cell associated with each DU-145 cell was ( 2 .0 6
O. 2 4 )x 1 04 for
TGS-GN P s, a n d (6.73 0.67)x 104 for Glu-GNPs (Fig. 2). In contrast,
exposure to Glu-GNPs results in
a three times increase of nanoparticle uptake compared to TGS-GNPs in each
target cell (Fig. 2). P
<0.005 for Glu-GNPs vs. TGS-GNPs (t test).
= Distribution of Glu-GNPs in DU-145 Cells
The distribution of GNPs in DU-145 cells was determined by TEM. Fig. 3 is the
micrograph
of the cells treated with 15nM Glu-GNPs. The figure shows that most of Glu-
GNPs were distributed
in the cytoplasm.
Effect of Gold-particles on DU-145 cell growth
Compared to the control, the results in Fig.4 show that DU-145 cell growth was
decreased by
13.52% with TGS-GNPs and17.82% with Glu-GNP treatments (P<0.01) after 24
hours. However,
there was no difference on cell growth between the TGS-GNPs and
Effect of 2 Gv 200 kVp X-rav on DU-145 cell viability
16
CA 02660507 2009-03-27
The cytotoxic effects of X-ray on DU-145 cells were analyzed after 24, 48 and
72 hours of
irradiation. Untreated control samples were arbitrarily assigned a value of
100% and the results of
all treatments were normalized to 100% (i.e., % of control). The data in Fig.
4 shows that 2 Gy X-ray
induced an inhibition of cell growth by 15.78%, 19.03% and 9.22% at 24, 48 and
72 hours respectively.
Gold-oarticles enhance radiation cvtotoxicitv on DU-145 cell
To determine whether GNPs had enhanced radio sensitivity of DU 145 cells to 2
Gy X-ray, cells
were treated with either a single dose of 2Gy X-ray or 2Gy X-ray and GNPs,
whereas control group did not
receive any treatment. Untreated control samples were arbitrarily assigned a
value of 100% and the
results of all treatments were normalized to 100% (i.e., % of control). Fig,
5A shows that either TGS-GNPs
or X-ray induced an inhibition of cell growth by 13.52% or 15.88% at 24 h,
individually. However, a
combination of TGS-GNPs and X-ray induced an inhibition of cell growth of
30.57% (P<0.005) (Fig. SA).
Similarly, the data in Fig. 5B shows that Glu-GNPs induced an inhibition of
cell growth by 17.82% after 24
hours but the combination of Glu-GNPs plus X-ray induced an inhibition of cell
growth by 45.97%
(P<0.005).
Enhancement of radiosensitivitv by either TGS-GNPs or Glu-GNPs
To evaluate whether glucose will help the delivery of gold-nanoparticles to
cancer cells, the
cellular uptakes (Fig. 2) and radiosensitivity enhancement induced by Glu-GNPs
were determined and
compared to those induced by TGS-GNPs. Fig. 6 shows that the inhibition rate
of 2Gy X-ray plus TGS-GNPs
was (30.57 3.32)% at 24 hours and (32.18 2.12)% at 48 hours. The inhibition
rate of 2Gy X-ray plus Glu-
GNPs was (45.97 3.95)% at 24 hours and (44.63 1.87)% at 48 hours. Glu-GNPs
increased radiosensitivity
by 50.37% (P < 0.001) at 24 hours and 38.68% (P <0.005) at 48 hours compared
with TGS-GNPs that have
no glucose bound.
Example Two ¨ Enhancement of Radiation Cvtotoxicitv in Breast Cancer Cells bv
Localized Attachment
of Gold Nanoparticles
Materials
All chemicals were obtained from Sigma¨Aldrich (Milwaukee, WI). MIT cell
proliferation assay kit
=
was purchased from Invitrogen (Burlington, Ontario).
Synthesis of Gold Nanooarticles
17
CA 02660507 2009-03-27
The general synthesis method for making gold nanoparticles followed three sub-
steps. i) 2m1 of
25mM HAuCts solution was added into 25m1 of deionized water in an ice bath
with moderate stirring. ii)
2m1 of 30mM NaBH4 was then added as a reductant to obtain GNPs without any
capping agents_ iii) To
functionalize the GNPs, 4m1 of 25mM thio-glucose or AET was added into the
previous gold solution,
respectively, to obtain functional gold nanoparticles. Considering the gold
nanoparticles in step (ii) are
easy to aggregate, sodium citrate (TGS) was added to cap them as naked gold
nanoparticles. The purpose
for using the same GNP solution was to ensure the resulting functionalized
nanoparticles had identical
sizes. Both AET-capped gold nanoparticles (AET-GNPs) and thioglucose capped
gold nanoparticles (Glu-
GNPs) were dialysed for two days before cell uptake and irradiation
experiments. The average sizes of
three types of GNPs (naked GNPs, AET-GNPs and Glu-GNPs) measured by dynamic
light scattering (DLS)
were about 10.8 nm. The surface of AET-GNPs and GiuGNPs are characterized by
using X-ray
Photoelectron Spectroscopy (XPS) (Kratos Analytical).
Cell culture and culture conditions
Human breast adenocarcinoma line MCF-7 was used in all the experiments. MCF-7
cell line was
purchased from American Type Culture Collection (Manassas, VA, USA). MCF-7
cells were maintained in
Dulbecco's Modified Eagle Medium (DMEM, lnvitrogen) containing 10% FBS
(Invitrogen), 100 Ul penicillin
G and 100 pg/m1 streptomycin (Sigma). The cells were incubated at 37c in a
humidified 5% CO2
atmosphere.
Cellular morphology with transmission electron microscopy
The cell cultures both treated with and without GNPs for two hours were
centrifuged and the
supernatants were removed. The pellets with 2 PBS washes were fixed in 4%
(v/v) formaldehyde in 0.1M
phosphate buffer (pH 7.2), for two hours at rc. After being washed in the same
buffer, the cells were
resuspended in 1% 0s04 for one hour at room temperature. They were then washed
twice by
centrifugation and resuspension in distilled water. The final pellet was
resuspended in a small volume of
warm 2% (w/v) agarose, poured onto a glass slide, and allowed to cool. When
set, small pieces of gel
containing the cells were cut out and dehydrated through a graded series of
ethanol solutions. The pieces
were then embedded in epoxy resin, and thin sections were cut with an
ultramicrotome, stained with
uranyl acetate followed by lead citrate and examined in a Philips EM301
electron microscope operating at
80 kV.
Determination of GNPs bound to or uptaken by target cells
A 5m1MCF-7 cell suspension containing 5 x105 cells was added to 6cm-dishes and
cultured
overnight. When the cells reached 70% confluence, the target cells were
exposed to 5 ml of fresh medium
18
CA 02660507 2009-03-27
with 15nM of GNPs (final concentration). After two hours of incubation, the
medium with GNPs was
removed and cells were washed twice with 5 ml PBS buffer. The cells were
detached with Trypsin/EDTA.
After centrifugation and removal of the supernatant, the cells were
resuspended into PBS to a final
volume of 5m1. The number of cells in suspension was counted with a
hemocytometer. 5m1 of 20% HNO3
was added into each sample to lyse the cells. The gold mass in the lysis
solution was measured by
Inductively Coupled Plasma Mass Spectrometry (ICP-MS). We can calculate the
number of gold
nanoparticles via the gold mass, and the number of GNPs in the lysis solution
divided by the number of
cells provided a quantitative measure of GNP cell uptake.
MTT assay and clonogenic survival assay
The cytotoxicity induced by GNPs with and without radiation was assessed by an
MTT assay in
96-well plates and clonogenic survival assay. MCF-7 cells were tested. The
starting number of cells was
3000 per well with 1501.11of medium. The cells were allowed to grow on the 96-
well plates overnight.
After 70% confluence was reached, the old medium was replaced with 150 pl of
freshly prepared medium
containing GNPs at the desired concentrations. After two hours of incubation
and the cell uptakes
4
reached about 2.96 x 10 GNPs per cell, the medium was replaced with fresh
medium. The cells were
treated with or without irradiation. Cell response to the GNPs with and
without irradiation was monitored
by the MTT assay following the manufacturer's instructions and clonogenic
survival assay. For clonogenic
survival assay, the cells either with or without treatment were incubated for
2-3 weeks. Cells were then
fixed with 3:1 ethanol to acetic acid solution and stained with crystal
violet. Colonies were counted for the
control and experimental groups, with each experiment performed in triplicate.
Cell Irradiation
Human breast cancer MCF-7 cells and non-malignant breast cell line (MCF-10A)
were used in
these studies. 200kVp X-ray irradiation with the PANTAK Therapax3 series, y-
ray irradiation with the
Shepherd-Mark I-68A 137Cs Irradiator(J. L Shepherd & Associates, San Fernando,
CA) and the 83Co
irradiator (Atomic Energy of Canada Ltd) were used in this experiment. The
targeted cells were exposed to
hybrid-gold nanoparticles (Glu-GNPs or AET-GNPs) and then followed by either
(I) 200kVp X-ray irradiation
at 1.19Gy/min, (ii) y-ray irradiation with I37Cs at ¨1.57 Gy/min, or (iii)
with 6 Co irradiator at ¨4.76 Gy/min,
each with a total dose of 10 Gy at room temperature. The control experiments
were performed with the
same procedures as above except replacing hybrid nanoparticles with non-
labeled naked GNPs. For all the
experiments, after irradiation, the cells were incubated for 48 hours before
measuring cell viability with
the MTT assay. The cells growing in plain medium without nanoparticles or
irradiation were used as a
control.
19
CA 02660507 2009-03-27
Statistical analysis
Experimental values were determined in triplicate. All values regarding
measurement and
percentage of gold content were expressed as means and standard errors (SE).
The one-way analysis of
variance (ANOVA) and Tukey multiple comparison post-test were used.
Differences less than 0.05 (p<0.05)
were considered statistically significant.
Characteristics of GNPs =
Figure 7 shows the schematic diagram of both AET-GNPs and Glu-GNPs. Figure 8
shows the TEM
images of the gold nanoparticles. The diameters of the GNPs were measured with
TEM and the average
diameter of GNPs was 10.8 nm. The same size GNPs were used for all experiments
in this study. To
identify the binding of GNPs with either AET or thioglucose, the nanoparticles
were characterized using
XPS. The average numbers of bio-molecules (around 2x one nanoparticle were
calculated by measuring
the gold to sulfur atom ratio acquired with XPS (Figure 9). The results are
listed in Table 1.
Table 1: Measurement results of gold and sulfur concentration
on AET-GNP and Glu-GNP.
AET-GNP Glu-GNP
Au (at. %) 64.55 59.76
S (at. %) 35.45 40.24
Distribution of GNPs in Targeted Cells
The typical distribution of GNPs in MCF-7 cells can be observed by TEM. Figure
10Mndicates the
appearance of normal cells. Figure 10C is the micrograph of the cells treated
with 15nM Glu-GNPs. The
figure shows that most of GNPs were distributed in the cytoplasm. The
appearance of MCF-7 cells treated
with 15 nM AET-GNPs is shown in Figure 10B, in which we can see that most GNPs
were bound to the cell
membrane in this case.
GNPs Bound to or Internalized bv Target Cells
The number of GNPs that bound to or were taken up by MCF-7 cells in cell
lysate was quantified
with ICP-MS. After the cells were exposed to 15 nM of naked GNPs, AET-GNPs, or
Glu-GNPs for two hours,
the average number of GNPs associated with each MCF-7 cell was 7.34 x 103 for
naked GNPs, 2.96 x 104
for Glu-GNPs, and 1.187x105 for AET-GNPs per cell (Figure 11). Exposure to AET-
GNPs resulted in a factor
of three to four-fold increase of GNPs compared to Glu-GNPs in each target
cell. When 15 nM Glu-GNPs
and 3.85 nM AET-GNPs were used to treat the cells respectively, the targeted
cells have the same level of
nanoparticles for each type of GNPs in the targeted cells, or about 2.96 x 104
GNPs per cell. We later use
these samples in the experiments of cytotoxicity assay and irradiation
studies.
CA 02660507 2009-03-27
Cvtotoxicitv of GNPs
The cytotoxicity of either AET-GNPs or Glu-GNPs on MCF-7 cells that were
treated for 24, 48, or
72 hours respectively was measured with the MTT assay. The data was analyzed
with a t-test, and no
significant difference was noticed between the control and the AET-GNP-treated
cells or the Glu-GNP -
treated cells (p<0.05) as shown in Figure 12. By using the percentage to
measure the viability, no
significant changes in the cytotoxicity were seen as incubation time
increased. Therefore, we can conclude
that neither the AET-GNPs nor the Glu-GNPs induced remarkable cytotoxicity in
MCF-7 cells.
Irradiation: Enhancement of irradiation bv GNPs
The target cells were treated with GNPs for two hours. Both the treated and
untreated cells were
then irradiated with 200kVp X-rays with a dose of 10Gy. We detected the
induced cytotoxicity with the
MTT assay 48 hours after the irradiation. The responses of the MCF-7 cell line
with and without X-ray
treatment are shown in Figure 13. After irradiation, 200 kVp stimulated cancer
cell growth and induced a
cell viability of 114.8% after 48 hours (Figure 13A). Glu-GNPs (15 nM) and AET-
GNPs (3.85 nM) induced
about 63.5% and 31.7% increase in radiation cytotoxicity respectively, when
compared to irradiation
alone.
In clonogenic survival assay, 200kVp X-rays induced a significant decrease in
cell survival (Figure
13B) after five days. However, there were still 43.2% of cancer cells
surviving after five days and 13.8% of
cells survived after fourteen days. For the cells exposed to Glu-GNPs, the
irradiation of 200kVp X-rays
resulted in complete cancer cell death after five days and this trend
continued.
=
Comparison of radiation sensitivity in cancer or non-malignant cells
MCF-10A cells were selected as a non-malignant breast cell line to provide
evidence to prove the
efficiency of the functional gold nanoparticles in cancer killings. After
exposing to Glu-GNPs, both MCF-7
and MCF-10A cells uptook the same level of GNPs because both types of cell
lines have the same growth
rates and need the same amount of glucose for metabolism (the results were not
shown in this paper).
Figure 14A shows that 200 kVp X-ray induced a 20% cell viability decrease in
the MCF-10A cells, but not in
the MCF-7 cells. After incubation with Glu-GNPs for two hours, the cell
viability of MCF-7 cells decreased
to 40% after irradiation. However, no significant changes in radiation
sensitivity were shown in MCF-10A
cells that were treated either with or without Glu-GNPs (p<0.05). These
results indicate that Giu-GNPs
only enhance the radiation sensitivity in cancer cells but not in non-
malignant breast cells. These results
also show that the modified nanoparticles of the invention can be used for
targeted cancer treatment.
21
CA 02660507 2009-03-27
Radiation cvtotoxicity resulting from various types of irradiation
The capability of GNPs to enhance the cytotoxicity induced in MCF-7 breast
cancer line by various
types of irradiation (X-ray and y-ray) was evaluated. Compared to 200kVp X-
rays (30% cell death for AET-
GNPs and 60% cell death for Glu-GNPs), 137Cs y-ray and 6 Co y-ray have smaller
enhancement on cell killing
(12.7% cell death forinCs y-ray and 13.1% for 6 Co y-ray) (Fig.148).
Example Three ¨ Making Gold Nanoparticles bound with PET Tracer
(118F1flurodeoxvglucose)
Step 1. Creating gold-based hybrid nanoparticles.
Step 2. Binding the hybrid nanoparticles covalently to PET tracers.
Step 3. Testing cell uptake of gold-based hybrid nanoparticles.
18F-6-FDG was synthesized using radioactively labeled fluorine ion as a
nucleophile for
displacement of a tosyl or trifyl group at C-6 of acetyl-protected glucose.
Ac0 Ac
1,2,3,4-Tetra-0-acetyl-beta-D-glucopyranose
For the organic synthesis of 18F-6-FDG capped gold nanoparticles, tosylation
or trifylation of the free
hydroxyl group at the C-6 position of 1,2,3,4-Tetra-0-acetyl-beta-D-
glucopyranose was done (shown in Fig.
19), a commercially available reagent from Sigma-Aldrich. Thioacetate was then
successfully displaced the
0-acetyl group at the C-1 position of the tosylate or trifylate intermediate.
After displacement of the tosyl
or trifyl by 18F, the radioactively labeled intermediate was hydrolyzed to
give a 18F labeled thioglucose,
which then binds to gold nanoparticle rapidly via a strong covalent bond
between the free thiol group and
gold nanoparticle, and subsequently ready for injection to patients. Both
2,3,4-Tri-O-acetyl-1-S-acetyl-1-
thio-6-0-(4-methylphenylsulforiy1)-beta-D-glucopyranose and 2,3,4-Tri-O-acetyl-
1-5-acetyl-1-thio-6-0-
trifluoro-methanesulfonyl-beta-D-glucopyranose were synthesized, which were
labeled with 18F (our final
as product shown in Fig. 20).
H H sH
FOG with thiol group, which is ready to bind with gold nanoparticle as shown
in Fig. 15.
22
CA 02660507 2009-03-27
Example Four ¨ FDG-Gold Nanoparticles Used as a Sensitizer of Radiotherapy in
vivo
Mouse Preparation
Approximately 5 week-old female Balb/C nude mice were used. All mice were
quarantined and
acclimatized to laboratory conditions for two weeks before inoculation with
tumour cells.
Cell Preparation
MCF-7 breast cancer cells were cultured until the mice were ready for
inoculation. The cells were
harvested, counted, diluted in 0.9% saline solution for injection.
Experimental Procedure
1) 200 1 of the above cell solution containing 5.0 X 106 MCF-7 cells was
injected subcutaneously
into the right flank of each mouse. Tumor volume was calculated from external
measurements of length
(L), width (W), and height (H) three times a week after tumor cell
inoculation. Calculation of tumor
volumes was based on the assumption that tumors will be hemi-ellipsoids:
V=L*W*H* 0.5236.
2) After tumor volumes reach about 300mm3, mice were ready for treatment and
were randomly
divided into 4 groups:
Group 1: Control ¨ tumour-bearing mice;
Group 2: GNPs ¨tumour-bearing mice injected FDG-GNPs;
Group 3: X-Ray ¨tumour-bearing mice treated by x-ray irradiation ; and
Group 4: X-Ray + GNPs ¨ tumour-bearing mice treated by x-ray irradiation after
FDG-GNPs
injection.
3) For groups 2 and 4, two hours before FDG-GNPs injection, food was withheld
from the mice.
200uL of FDG-GNPs solution suspended in sterilized RODI water was injected
into the tail vein of each
mouse.
4) For groups 3 and 4, x-ray irradiation was delivered at 2 hours post-
injection of FDG-GNPs (X-
Ray equipment: Pantek orth-voltage, 200Kvp, 10Gy).
5) Treatment of mice with FDG-GNPs injection and x-ray irradiation was done
weekly for four
straight weeks.
6) Mice were monitored daily for animal survival and morbidity and for weight
loss and inactivity
in addition to signs, such as scruffy appearance, listlessness, and
compromised breathing during the study
period. Meanwhile, tumor volumes were measured three times weekly.
End Point Determination
23
CA 02660507 2009-03-27
Mice were sacrificed when the tumor weight in the treatment and control groups
reaching 10%
of the baseline body weight, or when the largest tumor diameter reached 25mm,
or when the largest
tumor bi-dimensions reached 16mm x 16mm, which ever came first.
Data Analysis
All the data were normalized based on the tumor volume measurement prior to
first treatment
delivery. Data was analyzed using a one-way ANOVA test.
FDG-GNPs Sensitize Tumors to Irradiation Therapy in vivo
The difference between the group treated with irradiation only (-= -) and the
group treated
with irradiation plus FDG-GNPs (-v-) was significant (p = 0.00971), which
means FDG-GNPs
enhance radiosensitivity of breast cancer MCF-7 cell tumors to X-Ray
irradiation (200Kvp,
10Gy) in vivo in a mouse model.
Example Five ¨ TestinR Safety of PET-Guided Gold Nanoparticles in an Animal
Model
The acute toxicity of PET-guided gold nanoparticles will be studied in the
BALB/c mouse model. The
starting dose will be one-tenth of the safe dose identified in the mouse model
for the phase I clinical trial, which in
turn identifies human toxicity as the primary endpoint. There will be a dose-
escalation schedule within the human
trial. The acute toxicity of the nanoparticles will be assessed as per the
Organization for Economic Co-operation
and Development (OECD) guideline, with the Up-and-Down-Procedure (UDP) (#425).
The following is a detailed technical description of the in vivo test: four to
five weeks old BALB/c nude
mice will undergo tail-vein intravenous injection of gold nanoparticle fluid.
A total of 45 animals will be used in this
part of the study, 15 for PET-guided gold nanoparticles, 15 for regular gold
nanoparticles, and 15 for the control
group, in which saline alone will be injected intravenously. The control group
would account for the effect of
anesthesia and injection procedures. Gold nanoparticles at a starting
concentration of 100 ps per ml will be used
for each fixed volume (0.2 ml) of intravenous injection, as previous
experiments showed that this dose was not
toxic. On the first day, a concentration of 100 pg per ml of gold
nanoparticles and PET-guided nanoparticles will be
administered for one mouse from each group. The same volume of saline will be
injected in the control mouse. The
treated mice will be observed continuously for the first 4 hours, every hour
for the following 4 hours, then every 12
hours for the following 2 days, and then daily for 2 weeks.
24
CA 02660507 2015-10-07
The animals' morbidity will be assessed using a morbidity score. The animal
should be humanely
euthanized with euthanyl when the score indicates that the animal has reached
the end point. If the first
treated animals survive the concentration of 100 lig per ml during the first
24 hours, the next concentration
(1000m per ml) will be applied and the observations will be conducted as
previously mentioned. This will
provide a ten-fold dosing range to determine if the previously found
concentration is truly the most
appropriate dose without reaching toxic drug levels (high dose) or therapeutic
insensitive doses (low dose).
Upon reaching a concentration that is not successful, the dose will be
decreased to the last found
concentration. The previous procedures will be repeated until one of the
stopping criteria is satisfied. It is
expected that the reachable concentration will be over 1g per ml with the
soluble PET-guided nanoparticles.
Necropsy: All of the experimental mice will be sacrificed for necropsy.
Histological examination and
pathology reports will be issued by a consulting veterinarian. The liver,
lung, heart, kidney and brain will be
harvested from euthanized animals, and sent for examinations and nanoparticles
quantification. Blood will be
collected, using the cardiac bleeding technique, and sent for complete
chemistry panel and blood count.
ALTHOUGH PREFERRED EMBODIMENTS OF THE INVENTION HAVE BEEN DESCRIBED HEREIN IN
DETAIL, IT WILL BE
UNDERSTOOD BY THOSE SKILLED IN THE ART THAT VARIATIONS MAY BE MADE THERETO
WITHOUT DEPARTING FROM THE
SCOPE OF THE APPENDED CLAIMS.
